Development of Hydrogen Sulfide-Releasing Carbonic Anhydrases IX- and XII-Selective Inhibitors with Enhanced Antihyperalgesic Action in a Rat Model of Arthritis

An effective therapeutic approach based on the anti-inflammatory action of hydrogen sulfide (H2S) and inhibition of carbonic anhydrases (CAs) IX and XII is proposed here for the management of arthritis. H2S is a human gasotransmitter that modulates inflammatory response at low concentrations. Inhibition of CAs IX and XII can repristinate normal pH in the acidic inflamed synovial fluid, alleviating arthritis symptoms. We report here the design of H2S donor—CA inhibitor (CAI) hybrid derivatives. The latter were tested in vitro as inhibitors of human CAs I, II, IV, IX, and XII, showing a markedly increased inhibition potency/isoform selectivity compared to the CAI synthetic precursors. The best compounds demonstrated the ability to consistently release H2S and produce a potent pain-relieving effect in a rat model of arthritis. Compound 26 completely reverted the pain state 45 min after administration with enhanced antihyperalgesic effect in vivo compared to the single H2S donor, CAI fragment, or their co-administration.


■ INTRODUCTION
Rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) are inflammatory diseases affecting people over sixty and under sixteen years old, respectively, characterized by chronic inflammation of the synovial membrane, that leads to articular cartilage and juxta-articular bone destruction and, eventually, deformity. Both conditions are accompanied by pain. 1−3 The pathophysiology of RA and JIA is unknown to date but was shown to be related to the previous activation of endothelial cells with the new blood vessel growth and a hyperplastic expansion of the synovial membrane that invades the periarticular bone at the cartilage−bone junction, leading to bone erosions and cartilage degradation. 1−3 This process promotes the migration in loco of macrophages and leukocytes (T cells, B cells, and monocytes) that release pro-inflammatory cytokines, such as the tumor necrosis factor and interleukin-6 (IL-6), stimulating the production of several secondary modulators: the nuclear factor κB ligand (RANKL), which is responsible for the osteoclast differentiation and osteoblasts apoptosis, prostaglandins, and matrix metalloproteinases, that mediate the symptoms of the disease including pain, swelling, and degradation of cartilage and bone. 1, 2,4 An important piece of evidence is that the patients with RA showed the concomitant presence of a lower pH (6.5) in the synovial fluid compared to healthy patients or affected by osteoporosis, which is linked to inflammatory reaction and direct damage to the cartilage and tissue around the joint. 5−8 Furthermore, it was demonstrated that the synovial fluid becomes acidic as the intensity of the inflammatory response increases, and the more intense the inflammatory reaction was the more acidic the synovial fluid pH became, provoking pain. 5 Several isoforms of the metalloenzymes carbonic anhydrases (Cas and EC 4.2.1.1), that catalyzed the reversible hydration of carbon dioxide into bicarbonate and proton ions, have been associated with articular inflammatory diseases. 9, 10 In detail, CA I was found to be overexpressed in the synovium of the patients with ankylosing spondylitis, and transgenic mice that overexpressed CA I showed aggravated joint inflammation and destruction. 11,12 Furthermore, antibodies to CA III and IV have been identified in RA. 8 The latter study also showed that CA activity in RA was significantly higher than that of control groups. Most importantly, in 2016, Cimaz et al. demonstrated that isoforms CA IX and XII, which are usually related to hypoxic tumors, are also overexpressed in the inflamed synovium of patients affected by JIA. 13 Hydrogen sulfide is the third gasotransmitter of the human body, along with nitric oxide (NO) and carbon monoxide (CO), which have been widely investigated for biomedical purposes over the last three decades. 14 Its endogenous synthesis is attributed to three enzymes with different subcellular and tissue distribution called cystathionine-βsynthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, which use L-cysteine, L-homocysteine, and 3mercaptopyruvate, respectively, as the substrate. 14−17 Once synthesized by a cell, this ubiquitous small gaseous signaling molecule performs a paracrine action that involves at least 200 neighboring cells, thanks to its lipophilic nature which allows it to cross the membranes. 18 Hydrogen sulfide has not a specific receptor or signaling pathway, but it triggers many cellular effectors in a cell/tissue/species-dependent way, being involved in various and important physiological processes such as modulation of vascular tone and blood pressure, neurotransmission and nociception, angiogenesis, cardiac function, various leukocytic functions, penile erectile function, scavenger action, acting as an adenosine 5′-triphosphate (ATP) synthesis stimulator, and so forth. 14,18 Numerous studies demonstrated that the pathogenesis of many diseases, including several inflammatory disorders, is related to hydrogen sulfide deficiency, and the treatment with H 2 S-donating molecules improves symptoms. 14,19−28 Based on this evidence, hydrogen sulfide donor�CA inhibitor hybrid derivatives are here proposed to combine the action of H 2 S and inhibitors of specific human (h) CA isoforms for the treatment of inflammation, specifically RA. In fact, inhibition of the hCA isoforms, whose activity is abnormal on the synovial membrane, was shown to repristinate the normal pH of the synovial fluid, alleviating the RA and JIA symptoms (pain, cartilages, and bony erosion). 29 −33 On the other hand, the released H 2 S suppresses the inflammatory process on multiple levels; improves symptoms such as redness, swelling, heat, pain, and loss of function, blocking leucocyte adhesion, and promoting neutrophils apoptosis, analgesia, and reparation of the damage. 14,19 Moreover, H 2 S prevents the formation of the complex RANKL-RANK (ligand−receptor activator of nuclear factor kappa-Β), blocking the transcription of pro-inflammatory cytokines, osteoclast differentiation, and osteoblast apoptosis, protecting bone erosion. 4,22,34 A similar study was recently reported by some of us over the development of effective CAIs incorporating scaffolds that release carbon monoxide (based on the dicobalt hexacarbonyl complex), another gasotransmitter showing pharmacological application for the management of inflammation. 30 ■ RESULTS AND DISCUSSION Drug Design and Chemistry. The design of H 2 S donor� CAI hybrids took into account that H 2 S is also a rather toxic gas. In fact, if inhaled at concentrations of >20 ppm, it enters the bloodstream and tissues determining the blockage of the cell respiratory chain by binding the cytochrome C oxidase iron, inducing oxidative stress, and blocking the synthesis of ATP, which is fatal at a concentration of 1000 ppm. 15 For these reasons, it is important to control the H 2 S release in order to achieve an optimal concentration for the therapeutic effect and not poisoning. Among the numerous H 2 S-releasing scaffolds reported in the literature, which showed different gasotransmitter releasing properties, the drug design strategy here proposed was initiated with the 4-substituted 3H-1,2dithiole-3-thione chemotype (DTT, Figure 1), 35 chosen on the basis of the target therapeutic action and resulting necessary H 2 S release rate. As for including CAI scaffolds into the molecular hybrids, the choice fell on the benzenesulfonamide chemotype which furnishes optimal hCA inhibition potency and was shown to be suitable for drug design strategies aimed at enhancing isoform selectivity. 36,37 The synthesis of the H 2 S releaser�CAI hybrids starts with the preparation of the 3H-1,2-dithiole-3-thione core, which bears a p-COOH-phenyl moiety at position 4 for the subsequent hybridization with the CAI scaffold. Initially, 4isopropylbenzoic acid was protected as ethyl ester 1, using SOCl 2 in dry ethanol (EtOH) (Scheme 1). The cyclization to 3H-1,2-dithiole-3-thione was obtained by treating derivative 1 in melted sulfur at 220°C, to give intermediate 2, whose ester group was thus hydrolyzed with H 2 SO 4 in CH 3 COOH at 100°C , yielding the carboxylic acid 3. The coupling reaction of 3 with primary amine derivatives of benzenesulfonamides [e.g. sulfanilamide, 4-(aminomethyl)benzenesulfonamide, 4-(2-aminoethyl)benzenesulfonamide] with several coupling agents and reaction conditions unexpectedly always led to the undesired attack on the scaffold thione group with the formation of the imine-like derivatives A−C even at very low temperatures (Scheme 2).
As a result, it was supposed that the use of secondary amines benzenesulfonamide derivatives, such as 4−18, could prevent the formation of these undesired side products in the coupling reaction conditions used in our experiments (Scheme 3). Hence, 4-(2-aminoethyl)benzenesulfonamide was treated by reductive amination in presence of the proper aromatic aldehydes and sodium borohydride in dry methanol (MeOH) or, alternatively, by nucleophilic substitution with the appropriate halides in dry dimethylformamide (DMF), to give secondary amines 4−18. The latter was coupled with carboxylic acid 3 by using PyBOP as the coupling agent and DIPEA as a base in dry DMF at room temperature (r.t.).
CA Inhibition. The synthesized compounds 19−33 were tested for their inhibitory action against hCA isoforms I, II, IV, IX, and XII, by a stopped-flow kinetic assay using acetazolamide (AAZ) as standard. 38 The cytosolic hCA I and II are considered off-target for the CAI anti-inflammatory therapeutic application because they are ubiquitous and responsible for most CAI side effects. In contrast, hCAs IV, IX, and XII were the target isoforms being overexpressed on the synovial membrane and linked to inflamed conditions. [29][30][31][32][33]39 Table 1 gathers the inhibition constants (K I s) of compounds 19−33 against the panel of hCAs, whereas the selectivity index (SI) against hCA IV, IX, and XII over the off-target isoforms hCA I and II are collected in Table 2.
Most tested compounds displayed weak inhibition against the off-target hCA I, with K I values in a rather flat micromolar range (K I s of 2215−10000 μM), except derivatives 19 and 28 that inhibited this ubiquitous isoform in the high nanomolar range (K I of 770.7−890.3 nM). In contrast, all tested compounds produced a greater hCA II inhibition when compared to hCAI (K I s in the range 218.5−4826 nM), with the exception of compound 29. In fact, derivatives 19, 20, 27, 28, and 32 inhibit hCA II significantly below the micromolar range (K I s of 218.5−660.9 nM). It should be noted that the elongation of the N-substituent from a benzyl (19) to a phenethyl (32) decreases significantly the inhibitory action against hCA I (K I from 770.7 to 4387 nM) and with minor extent against hCA II (K I from 479.9 to 660.9 nM). Interestingly, the only tertiary amide derivative not bearing an aromatic scaffold on the N-substituent, that is the nitrile 33 (R = CH 2 CN), shows the greatest efficacy against hCAs I and II (K I s of 429.2 and 60.4 nM, respectively). It can be speculated that the loss of steric encumbrance of 33 in comparison to other derivatives favors the ligand binding in the narrow active sites of these ubiquitous isoforms. As a matter of fact, all substituents included on the outer phenyl ring of compound 19 decrease the inhibitory action against the cytosolic isoforms, with the only exception being the dimethoxy derivative 28 against hCA II (K I of 479.9 vs 218.5 nM).
As for hCA IV, the p-CN-phenyl and benzo[b]thiophen-3-yl derivatives 23 and 31 only exhibit K I values below 100 nM (K I s of 46.2 and 48.0 nM), resulting in the most potent and also the first and the second most selective compounds against hCA IV over hCA II (SI of 104 and 52, respectively).
Compounds 21, 28−30, 32, and 33 produce instead hCA IV inhibition in the high nanomolar range (K I s of 243.2−523.1 nM), while inhibitors 19, 22, 26, and 27 acted in the low micromolar span (K I s of 1042−4339 nM)and up to no inhibition detected below 10 μM for 20 and 25.
Notably, hCA IX and XII were the isoforms most inhibited by all 3H-1,2-dithiole-3-thione derivatives, with most K I s collected in a narrow range for both isoforms (K I s spanning between 2.4−50.3 nM and 5.7−32.9 nM for hCA IX and XII, respectively). It should be stressed that the target isozymes IX and XII show in fact rather roomier active sites when compared to other hCAs, which can favor the accommodation and binding of the bulky tertiary amide derivatives 19−32 after the binding of the sulfonamide moiety to the zinc ion. In particular, compounds 31 and 26 were the most potent and selective hCA IX inhibitors with a single digit (K I s of 2.4 and 4.1 nM, respectively, and hCA II/hCA IX SI of 1034 and 719, respectively). As with hCAs I and II, the elongation of the Nsubstituent from 19 to 32 led the K I above 100 nM (K I = 125.7 nM). All other hybrid compounds exhibited medium nanomolar range hCA IX inhibition with K I values between 15.6 and 50.3 nM.
On the other hand, hCA XII was inhibited with single-digit K I s by a greater subset of compounds (19,21,23, 24, 26, and Overall, the obligatory choice of coupling bulky secondary instead of primary amine benzenesulfonamide derivatives to the H 2 S donor nucleus, due to side products formation in the planned synthetic pathway, resulted to be a good option for the achievement of potent hCA IX and XII inhibitors also showing a high selectivity over off-target hCAs. In fact, the incorporation of N-alkyl aromatic substituents led the 3H-1,2-dithiole-3-thione derivatives to prefer the binding to the roomier active site of hCA IX and XII with respect to other hCA isoforms. In fact, it can be noted that the 2-ethylcyano derivative 33 showing the least steric encumbrance within the series exhibited a significant loss of target/off-target selectivity of action in comparison to most other such hybrid compounds, with the hCA II/IX SI being only 2.1.
It should be also stressed that the secondary amine benzenesulfonamide derivatives 4−18 do not show notable CA inhibition profiles both in terms of potency and isoform selectivity, as previously reported, 32,33 and here further investigated. Table S1, Supporting Information collects the inhibition profiles of 4−18 against hCA I, II, IV, IX, and XII, while the related SI values were reported in Table S2 . It is worth noting that, as previously shown, 35,36 most such single CAI derivatives did not show any relevant isoform selectivity, but instead exhibit a rather flat inhibition profile against target and off-target isozymes.
Evaluation of H 2 S Release. The H 2 S releasing properties of the 4-substituted-3H-1,2-dithiole-3-thione precursor 3 and    (Table S3, Supporting Information). The gasotransmitter release of the tested hybrid compounds overall reached a plateau after approximately 25 min. The H 2 S release profile of the DTT derivatives here tested is consistent with that of previously described similar such derivatives, although differences exist in the compound chemical structures and in the adopted spectrophotometric protocol. As a matter of fact, in 2007 Li et al. showed in vitro consistent H 2 S release profiles of the 5-substituted DTT compound ADTOH and its diclofenac derivative, when incubated in rat liver homogenate. 41 The same authors evaluated successively ADTOH and the 4-substituted analogue ACS48 (compound 3 in the present work) for their H 2 S donating properties in the cell, also showing a sustained release. 44 In 2017, Li et al. also evaluated, in vitro, a similar H 2 S release for ADTOH and some such nonsteroidal anti-inflammatory derivatives. 45 The authors deduced a hydrolytic trigger for the compound gasotransmitter release, but the reducing agent TECP, that is tris(2carboxyethyl)phosphine, was actually used. In 2018 Giustarini et al. also demonstrated the sustained H 2 S release of 3 (ACS48), in vivo, by monitoring the gasotransmitter concentration in plasma of rats treated intravenously with such a DTT and some of its derivatives. 35 As a result, to the best of our knowledge, the 4-substituted DDT 3 and amide derivatives, such as 19−33, were here assessed for the first time for their H 2 S release, in vitro.
In Vivo Studies. On the basis of the superimposable H 2 S releasing profiles of all tested hybrid derivatives, 26, 29, 30, and 31 were also evaluated for their anti-inflammatory effect in a rat model of arthritis induced by the intra-articular injection of complete Freund's adjuvant (CFA) in the Paw pressure and Incapacitance tests. 33 Moreover, the synthetic precursors 3, as the single H 2 S releasing molecule, and 15, as the most potent single CAI warhead against the target hCAs IX and XII, were tested to evaluate the hybrid efficacy with respect to the single agents, and their coadministration. The CFA model induces pain and articular damage characterized by diffuse lesions caused by an immune reaction towards antigens of connective tissue or joint. 46 The primary target of this response is the collagen that can be disrupted by T cells activation. The pathogenesis is characterized by augmented levels of proinflammatory cytokines, 47 neutrophil infiltration and subsequent synovia hyperplasia with a total ablation of the joint space that is replaced by hyperplastic tissue. 48 The pain-relieving properties of compounds were evaluated after a single per os administration in a dose ranging from 1 to 30 mg/kg when pain and articular lesions were well established (day 14 from the induction of the damage). The Paw pressure test was used to measure the hypersensitivity in response to a mechanical noxious stimulus ( Figure 3). In the group treated with CFA (red line), the weight tolerated on the ipsilateral paw decreased to 33 g in comparison to the control value of 65 g (black line) in the untreated rats (vehicle + vehicle). Compound 26 evoked an antihypersensitivity effect in a dose-dependent manner. In particular, the dose of 30 mg/kg counteracted the mechanical hyperalgesia 45 min after injection, showing a mild effect also at 60 min. The lower doses were still active and even showed a lower efficacy ( Figure  3A). Compounds 29, 30, and 31 were active in reducing the mechanical hyperalgesia evoked by CFA intra-articular (i.a.) injection with no dose-response effect between the dose of 10 and 30 mg/kg. Both dosages significantly increased the weight tolerated by the animals on the ipsilateral paw in comparison to CFA-treated animals starting from 15 min up to 45 min  Figure 3E reported the comparison between the antihyperalgesic effect evoked by hybrid 30 30 mg/kg in comparison to the equimolar doses of its single portions, that is the H 2 S donor 3 and the CAI 15, selected for being the most potent inhibitor of hCAs IX and XII among the secondary amine derivatives 4−18. The effect of 30 was also compared to the co-administration of 3 and 15. Despite 30 not standing as the best hybrid compound in terms of in vivo efficacy in the Paw pressure test, it clearly showed a greater antihyperalgesic action with respect to 3, 15, and their co-administration, highlighting the advantage of our strategy intended to merge in a single-molecular entity the two therapeutically active portions ( Figure 3E). Unlike the hybrid derivatives, the single H 2 S donor 3 evoked a rapid antihyperalgesic effect that peaked at 15 min and totally disappeared after 45 min. Such a trend might be partially justified by the greater H 2 S release rate detected for compound 3 ( Figure 2) in comparison to the other amide derivatives. Interestingly, compound 15, which possesses CA inhibition properties only, showed a very weak antihyperalgesic action at the dose here evaluated (17.71 mg/kg) equimolar to the active dose of the hybrid compounds (30 mg/kg). As a matter of fact, in the previous study regarding CO releaser�CAI hybrid compounds, a benzenesulfonamide CAI derivative inducing a potent inhibition of the target hCAs IX and XII, referred to as 3a, 30 showed a detectable pharmacological action in the Paw pressure test only when administered at a 4-fold higher molar dose than 15. The different dosages administered for 15 (present study) and 3a (past study) 30 depend on the 4-fold lower dose necessary for the best hybrid H 2 S releaser here reported (26) to achieve a pharmacological action even higher than the best hybrid CAI�CO releaser (referred as 5b) 30 in the Paw pressure test. In contrast to the rapid action of the H 2 S donor 3, the CAI 3a exhibited a peak of antihyperalgesic at 45 min. 30 To exclude possible toxicity induced by the acute treatment with the described hybrids, autonomic, behavioral, and neurological parameters were assessed by the Irwin test. As reported in Table S4, Supporting Information the highest dose of all compounds did not alter the endpoints analyzed.
The hybrid derivatives 26,29,30, and 31 were also tested against postural unbalance, a typical feature of monolateral pain, using the Incapacitance test as an evaluation of spontaneous pain since no direct noxious or non-noxious stimuli are applied by the operator (Figure 4). The difference between the weight bore on the contralateral and the ipsilateral paw was significantly increased up to 55 g (red line) in CFAtreated rats in comparison to the 2 g of the control group (black line). As in the Paw pressure test, similar results were obtained in this behavioral measurement. Compounds 26, 29, and 31 reduced spontaneous pain in a dose-dependent manner with a peak of efficacy between 30 and 45 min after treatment ( Figure 4A,B,D). Again, compound 26 demonstrated the greatest antihyperalgesic action almost completely reverting spontaneous pain after 45 min. Compound 30 oddly showed a slightly greater efficacy at 10 mg/kg in comparison to the dose of 30 mg/kg, while the lowest dose was ineffective ( Figure  4C). In the Incapacitance test, compound 30 induced a greater antihyperalgesic action than the co-administration of 3 and 15 ( Figure 4E). Nonetheless, in this test the action of compound 3 alone also exceeded that of the combination therapy and was comparable to that of the hybrid analogue 30 at its optimal dose ( Figure 4E). Again, the antihyperalgesic effect of the H 2 S releaser 3 rapidly peaked at 15 min, already disappearing after 30 min. Again, in absence of effects detected in vivo for 15 at the used dose, the CAI 3a previously reported 30 can be used to prove the antihyperalgesic action of a single CAI warhead in the Incapacitance test. In the latter, compound 3a, tested at a 4-fold greater dose than 15, exhibited a weak action with a peak at 45 min.
Relevantly, also in the Incapacitance test, the antihyperalgesic action of the best H 2 S releaser hybrid 26 significantly exceeded, according to a 4-fold lower administered dose (30 vs 100 mg/kg, and greater molecular weight), that induced by the best hybrid CAI�CO releaser (5b), which in turn was comparable to the reference drug ibuprofen 100 mg/kg. 30 In both in vivo tests, the antihyperalgesic action of the evaluated hybrid derivatives reached a maximum effect after 30 min post-administration with the exception of compound 26, which showed better efficacy and prolonged action over analogues 29−31. The H 2 S release of the hybrid derivatives, detected by a methylene blue assay in a sealed system, showed a plateau after 25 min approximately. In contrast, a more sustained H 2 S release was notified in vivo (open system) for similar such derivatives, 41 which might account for an antiinflammatory action peak in the range 30−45 min. On the other hand, it can be supposed that the trend of the hybrid in vivo action is consistent with a biological response being the result of the merged H 2 S release (peak at 15 min) and target CA inhibition (peak at 45 min as previously shown). 30 The detected enzymatic/metabolic H 2 S release trigger for the DTT scaffold, rather than hydrolysis and thiol activation, might be an advantage for the setup of such an antiinflammatory therapy. In fact, it can be supposed that the DTT derivatives start the gasotransmitter release only after absorption has occurred.
On the basis of the multitarget or hybridization approach here proposed, the exceptionally improved antihyperalgesic profile of the hybrid derivatives over the single agents and their co-administration could be rationalized as upgraded pharmacokinetics driving both pharmacophores (H 2 S donor and CAI), combined as a unique molecule, to the target inflamed site, where a strong synergistic effect takes place. Additionally, it can be speculated that the pharmacophore hybridization enhances drug absorption, which may be instead diminished in case of a coadministration.
■ CONCLUSIONS H 2 S is a ubiquitous small gaseous signaling molecule, playing an important role in many physio-pathological processes. For instance, H 2 S-donating molecules attracted enormous interest as they were able to promote the resolution of inflammation at the low endogenous gas concentration. On the other hand, some of us recently showed that a panel of CA isoforms, with CAs IX and XII as the main targets, are implicated in the pathogenesis of inflammation, and their inhibitors can repristinate the normal pH in the acidic inflamed synovial fluid, alleviating arthritis symptoms.
Hence, we described herein for the first time the synthesis and biological evaluation of new hybrid derivatives possessing a CAI warhead of the sulfonamide type conjugated to a DTT core as an H 2 S donating molecule. All hybrid compounds were tested in vitro as inhibitors of human (h) CA I, II, IV, IX, and XII, showing a marked increase in isoform-selectivity upon the proposed hybridization strategy over the CAI synthetic precursors. Compounds 26 and 31 were the most potent and selective hCA IX inhibitors with single-digit K I s of 4.1 and 2.4 nM, respectively, and hCA II/hCA IX SI of 1034 and 719, respectively. Also, 26 and 31 were among the best hCA XII inhibitors with K I s of 7.7 and 8.0 nM, respectively, and hCA II/hCA XII SI of 383 and 299, respectively. The best among such compounds, 26, 29, 30, and 31, along with the precursor and previously reported DTT 3, were shown to consistently release H 2 S by a spectrophotometric methylene blue method. Compounds 26, 29, 30, and 31 induced a pain-relieving effect in a rat model adjuvant-induced arthritis with a single i.a. injection of CFA. Compound 26 showed the greatest efficacy in vivo completely reverting pain 45 min after treatment both in the Paw pressure and Incapacitance tests. All tested compounds demonstrated a markedly greater antihyperalgesic action compared to treatment with the single H 2 S donor 3, the CAI fragment 15, and their co-administration. A comparison was thus made with previously reported CAI�CO releaser hybrid derivatives. 30 Relevantly, the performance of the best H 2 S donor hybrid 26 significantly exceeded, according to a 4fold lower administered dose (30 vs 100 mg/kg), that induced by the best hybrid CAI�CO releaser (5b), which in turn was comparable to the reference drug ibuprofen 100 mg/kg. Compound toxicity induced by the acute treatment was excluded from evaluating the animal autonomic, behavioral, and neurological parameters by the Irwin test. In conclusion, the hybridization drug design approach here proposed drastically improved the anti-inflammatory profile of the ■ EXPERIMENTAL SECTION Chemistry. Anhydrous solvents and all reagents were purchased from Sigma-Aldrich, Alfa Aesar, and TCI. All reactions involving airor moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic resonance ( 1 H NMR, 13 C NMR, 19 F NMR) spectra were recorded using a Bruker Advance III 400 MHz spectrometer in dimethylsulfoxide (DMSO)-d 6  General Synthetic Procedures for Secondary Amines 4−18. Procedure 1. The appropriate aldehyde (1.1 equiv) was added to a solution of 4-(2-aminoethyl)benzenesulfonamide (4,99 mmol, 1.0 equiv) in dry MeOH (20 mL), and the mixture was heated under stirring to reflux temperature for 0.5−4 h. Sodium borohydride (1.6 equiv) was added portion-wise at 0°C, and the reaction mixture was heated under stirring to reflux temperature for 0.5−3 h. The solvent was evaporated under a vacuum, water was added (25 mL) to it, and it was neutralized with HCl 1 M. The suspension was filtered, and the collected powder was occasionally purified with flash chromatography (MeOH 5%/DCM) to give the compounds 4−16.

N-(4-(Dimethylamino)benzyl)-N-(4-sulfamoylphenethyl)-4-(3thioxo-3H-1,2-dithiol-4-yl)benzamide (24).
Compound 24 was obtained as an orange powder according to the general procedure reported earlier using 9 (1.1 equiv) (25). Compound 25 was obtained as a yellow powder according to the general procedure reported earlier using 10 (1.1 equiv) (29). Compound 29 was obtained as an orange powder according to the general procedure reported earlier using 14 (1.1 equiv) CA Inhibition. An Applied Photophysics stopped-flow instrument has been used for assaying the CA-catalyzed CO 2 hydration activity. 38 Phenol red (at a concentration of 0.2 mM) has been used as an indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as a buffer and 20 mM Na 2 SO 4 (for maintaining constant the ionic strength), following the initial rates of the CAcatalyzed CO 2 hydration reaction for a period of 10−100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5−10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at r.t. before assay to allow for the formation of the E−I complex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng−Prusoff equation, as reported earlier, 39 and represent the mean from at least three different determinations. The enzyme concentrations were in the range of 3−11 nM. All hCA isoforms were recombinant ones obtained in-house, as reported earlier. 32,33 Spectrophotometric Evaluation of H 2 S Release. Rat liver was homogenized in ice-cold 50 mM potassium phosphate buffer (pH 7.4). 41 Optimal w/v ratios of 1/20 were determined from preliminary experiments (data not shown). The 3H-1,2-dithiole-3-thione derivatives were stocked as 30 mM solution in DMSO. The assay mixture (400 μL) contained the DTT compound incubated at 150 μM in phosphate buffer either in the presence or absence of a thiol derivative (such as L-cysteine and glutathione at 1.0 mM concentration) or in fresh rat liver homogenate (upon a further 1 to 10 dilution with phosphate buffer) in airtight vials at 37°C for timed intervals (0−90 min). The released H 2 S concentration was measured as described previously. 45 49 All efforts were made to minimize animal suffering and to reduce the number of animals used.

N-(4-(Methylthio)benzyl)-N-(4-sulfamoylphenethyl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)benzamide
Complete Freund's Adjuvant-Induced Arthritis. Articular damage was induced by injection of Complete Freund's Adjuvant (CFA; Sigma-Aldrich, St Louis, MO, USA), containing 1 mg/mL of heatkilled and dried Mycobacterium tuberculosis in paraffin oil and mannide monooleate, into the tibiotarsal joint. 50 Briefly, the rats were lightly anesthetized with 2% isoflurane, the left leg skin was sterilized with 75% ethyl alcohol, and the lateral malleolus was located by palpation. A 28-gauge needle was then inserted vertically to penetrate the skin and turned distally for insertion into the articular cavity at the gap between the tibiofibular and tarsal bone until a distinct loss of resistance was felt. A volume of 50 μL of CFA was then injected (day 0). Control rats received 50 μL of saline solution (day 0) in the tibiotarsal joint.
Administration of Compounds. The hybrid derivatives 26, 29, 30, and 31 (1, 10, and 30 mg/kg) were suspended in a 1% solution of carboxymethylcellulose (CMC) sodium salt and acutely per os administered starting from day 14 after CFA i.a. injection, when arthritis was well established. The H 2 S releaser 3 and CAI 15 were also administered alone or in coadministration at the equimolar dose of compound 30 30 mg/kg (13.23 and 17.71 mg/kg, respectively). The antihypersensitivity effects were evaluated over time by the Paw pressure and Incapacitance tests.
Paw Pressure Test. The nociceptive threshold of rats was determined with an analgesimeter (Ugo Basile, Varese, Italy), according to the method described by Leighton et al. 51 and Maresca et al., 52 (doi: 10.1111/jphp.12828). Briefly, a constantly increasing pressure was applied to a small area of the dorsal surface of the hind paw using a blunt conical probe by a mechanical device. Mechanical pressure was increased until vocalization or a withdrawal reflex occurred while rats were lightly restrained. Vocalization or withdrawal reflex thresholds were expressed in grams. Rats scoring below 40 g or over 75 g during the test before drug administration were rejected (25%).
Incapacitance Test. Weight-bearing changes were measured using an incapacitance apparatus (Linton Instrumentation, Norfolk, U.K.) to detect changes in postural equilibrium after a hind limb injury. 53,54 Rats were trained to stand on their hind paws in a box with an inclined plane (65°from horizontal). This box was placed above the incapacitance apparatus. This allowed us to independently measure the weight that the animal applied on each hind limb. The value reported for each animal is the mean of five consecutive measurements. In the absence of hind limb injury, rats applied an equal weight on both hind limbs, indicating postural equilibrium, whereas an unequal distribution of weight on the hind limbs indicated a monolateral decreased pain threshold. Data are expressed as the difference between the weight applied to the limb contralateral to the injury and the weight applied to the ipsilateral limb (Δ weight).
Toxicity and the Irwin Test. Toxicity was evaluated after a single acute administration of 26,29,30, and 31 at 30 mg/kg. Animals were observed for 24 h. For the Irwin test, each rat was individually placed in a transparent cage (26 × 41 cm), and 26 neurobehavioral or physiological parameters were systematically assessed according to Irwin (1968) 55 and already reported by Berrino and colleagues in similar studies. 30 Behavioral, autonomic, and neurological manifestations produced by compound administration in rats were evaluated: motor displacement, motor reflexes, stereotypies, grooming, reaction to painful or environmental stimuli (analgesia and irritability), startle response, secretions, excretions, respiratory movements, skin color and temperature, piloerection, exophthalmos, eyelid and corneal reflexes, muscle tone, ataxia, tremors, head twitches, jumps, convulsions, Straub tail, and other signs or symptoms. For postural reflexes (righting reflex) and other signs such as piloerection, exophthalmia (exaggerated protrusion of the eyeball), ataxia, tremors, and Straub tail, only presence or absence was recorded. Skin color was evaluated qualitatively (pale, red, or purple); other signs were evaluated semiquantitatively, according to the observer's personal scale (0 to +4, −4 to 0, or −4 to +4). The terms sedation and excitation express the final interpretation of a group of signs: reduced motor activity, reduced startle response, eyelid ptosis, and reduced response to manual manipulation, for the former; and increased motor activity, increased startle response, increased response to manual manipulation, and exophthalmia, for the latter. Hyperactivity includes running, jumping, and attempting to escape from the container. Trained observers were not informed about the specific treatment of each animal group carried out in this test.
Liver Collection. At the end of the behavioral measurements, control animals were sacrificed by decapitation. The liver was collected and immediately frozen in liquid nitrogen to determine the release of H 2 S by spectrophotometric analysis as described above.
Statistical Analysis. Behavioral measurements were performed on eight rats for each treatment carried out in two different experimental sets. Results were expressed as the mean (SEM) with a one-way analysis of variance. A Bonferroni's significant difference procedure was used as a post hoc comparison. P-values of <0.05 or <0.01 were considered significant. Data were analyzed using the Origin 9 software (OriginLab, Northampton, MA, USA).